The perfused small intestine from a vitamin D deficient rat exhibits about one-half the calcium transport of the intestine from a rat given vitamin D. These levels of calcium transport can be maintained for at least 4 hours. Addition of 2.5 micrograms of 25-hydroxycholecalciferol added to the vitamin D deficient intestine via the arterial blood perfusate induces a rise in calcium transport to +D levels within 2 hours. In contrast, 250 micrograms of vitamin D3 given in the same manner has no effect on the calcium transport level over a 4-hour period. These data provide strong evidence that 25-hydroxycholecalciferol represents the metabolically active form of vitamin D3.